Allium sphaeronixum (Amaryllidaceae), A New Species from Turkey

In this paper, Allium sphaeronixum, a new species of the sect. Codonoprasum from Turkey, is described and illustrated. The new species is endemic to Central Anatolia, limited to the area of Nevşehir, where it grows on sandy or rocky soil at an elevation of 1000–1300 m a.s.l. Its morphology, phenology, karyology, leaf anatomy, seed testa micromorphology, chorology, and conservation status are examined in detail. The taxonomic relationships with the closest allied species, A. staticiforme and A. myrianthum, are also highlighted and discussed.


Introduction
Allium L. is the largest genus of petaloid monocotyledons, comprising over 1100 accepted taxa [1]. It is represented by about 230 taxa in Turkey, grouped into 14 sections, with 86 endemics. In particular, the sect. Codonoprasum Rchb. is the second largest section in Turkey, with ca. 60 taxa, of which more than 30 are endemic [2][3][4].
During field surveys in the neighborhood of Nevşehir (Central Anatolia, Turkey), some populations of a very peculiar Allium clearly belonging to the sect. Codonoprasum were collected and investigated. Morphologically, these plants showed some resemblance with A. myrianthum Boiss., a species widespread in Anatolia, as well as with A. staticiforme Sm., found in the Aegean islands. According to [12], these species belong to a very peculiar group, with A. staticiforme as the most representative taxon, which is distributed in the East Mediterranean territories. The A. staticiforme group is well differentiated from all other known taxa of sect. Codonoprasum by having the following distinct selection of morphological traits: dense spherical inflorescence, with rather short spathe valves; small-sized perigon (2-4 mm long); stamen filaments, with all or at least the inner ones exserted; and seeds less than 4 mm long.
The aim of this study was to perform careful biosystematic analyses using living specimens cultivated both in the Botanical Garden of Catania (Italy) and in the Geophyte Garden in Yalova (Turkey) in order to clarify the taxonomic position of Nevşehir plants and their relationships within the staticiforme group. The performed investigations revealed that the populations from Nevşehir were closely related to A. myrianthum and A. staticiforme, which indeed clearly differ in many significant morphological features regarding the scape, number, and size of leaves; size and/or color of flower pieces (tepals, anthers, ovary, and style); and fruit size. A relevant diversity was also found in the karyotype structure, leaf anatomy, and seed microsculptures. Therefore, this Allium coming from Nevşehir is here treated as a new species for science, named A. sphaeronixum.
Etymology: The specific epithet, coming from the Latin words sphaera = ball and nix = snow, refers to the globose whitish inflorescence resembling a snowball (Figure 2A).
Suggested Turkish name: The Turkish name of this species is suggested as 'Kartopu Soganı' [13].
Phenology: The species flowers from July to mid-August, and fruit ripening occurs ca. one month after flowering.

Karyology
The somatic chromosome number of Allium sphaeronixum ( Figure 3A) was found to be 2n = 2x = 16 in all studied samples. The karyotype is rather asymmetrical, comprising only three fully metacentric pairs, while the remaining chromosomes include three meta-submetacentric pairs (arm ratio exceeding 1.30), one submetacentric and one subtelocentric pair, the last two pairs being, respectively, macro-and micro-satellited on the short arms ( Figure 4A). One small B-chromosome was detected in most of the observed metaphase plates. Thus, the chromosome formula of A. sphaeronixum can be expressed as 2n = 2x = 16 = 6 m + 6 msm + 2 sm sat + 2 st sat + 0-1B. The absolute chromosome length varied from 9.88 ± 0.6 µm of the longest chromosome to 5.31 ± 0.4 µm of the shortest one, with a mean chromosome length of 7.75 ± 1.4 µm. The relative chromosome length varied from 7.97% ± 0.3 to 4.28% ± 0.3. The arm index varied on average from 1.08 to 3.39, while the centromeric index ranged from 48.11 to 22.66, with a mean value of 39.3 ± 8.7.
As far as the two most allied species are concerned, the chromosome complements of both Allium myrianthum and A. staticiforme from their respective type localities were also examined for comparison ( Figure 3B,C). The karyotypes of these species appeared quite different from that of A. sphaeronixum, though sharing the same diploid (2n = 16) chromosome arrangement [12]. Karyotypes of A. staticiforme mostly showed more or less median chromosomes (five pairs fully metacentric and two pairs meta-submetacentric), while two chromosomes were submetacentric and microsatellited on the short arm, which can be determined by the following chromosome formula: 2n = 2x = 16: 8 m + 2 m sat + 2 msms + 2 msm sat + 2 sm sat ( Figure 4C). Conversely, A. myrianthum revealed a quite homogeneous karyotype, characterized by only metacentric chromosomes, two pairs of which were microsatellited on the short arm, with a chromosome formula 2n = 2x =16: 12 m + 4 m sat ( Figure 4B). All karyomorphometric parameters for the new species and its closest allied taxa are given in Table 2.

Leaf Anatomy
The leaf cross-section of Allium sphaeronixum showed a subcylindrical outline, with several inconspicuous ribs along the abaxial surface, while the adaxial one is flat to slightly concave and bordered by two evident ribs. The epidermis has small cells covered by a thin cuticle. Stomata are numerous and distributed along the whole leaf perimeter. The palisade tissue is compact and uniformly arranged in two layers of cylindrical cells, a little smaller in the adaxial face. The spongy tissue appeared distributed only in the peripherical part of the mesophyll, as the leaf is widely fistulous. Many secretory ducts occurred under the palisade tissue. There were about 8 big-sized vascular bundles in the abaxial part, alternating with. ca. 10 smaller ones, while 5 small vascular bundles occurred along the adaxial face (Figures 5A, S2A and S3A).   Conversely, the leaf cross sections of A. myrianthum ( Figures 5B, S2B and S3B) showed a semicylindrical outline with eight regularly prominent ribs with distinct hyaline tips. The epidermis consisted of large cells with a well-developed cuticle layer. The palisade tissue appeared two-layered, while the spongy tissue occurred only in the peripheral part because the mesophyll appeared widely fistulous in the center. Seven large vascular bundles occurred along the abaxial faces in correspondence with the ribs, while there were just three vascular bundles along the adaxial face.
As in A. myrianthum, the leaves in A. staticiforme also showed a semicylindrical outline ( Figures 5C, S2C and S3C), with 10-13 more or less prominent ribs along the abaxial surface, each ending with a small hyaline tip, while the adaxial face was smooth and distinctly concave. The epidermis showed small cells covered by a well-developed cuticle; the palisade tissue consisted of two layers of long cylindrical cells, while the spongy tissue appeared compact with small cells in the peripheral part, becoming looser with large cells in the center. There were 6-8 large-sized vascular bundles along the abaxial face alternating with 6 smaller ones, while only 5 small vascular bundles occurred in the adaxial face.
Overall, the seed testa micromorphology of A. sphaeronixum ( Figure 6A) reflected the main pattern already detected in other species of the sect. Codonoprasum [9,19,21,23,27]. The seeds were 3.5-3.7 × 2-2.2 mm in size, showing a semi-ovoid outline and a minutely papillate surface. At high magnifications (600× and 1200×), the testa cells revealed a subpolygonal and nearly isodiametric shape (21-36.5 × 16.5-32 µm), with minutely and irregularly undulate borders. The anticlinal walls appeared rather depressed and partly covered by the presence of feebly strip-like depositions forming an intercellular region 1.8-6 µm wide. The periclinal walls were slightly raised, usually provided with a large central and papillate verruca, surrounded by smaller peripheral ones, which made them very variable in number and size.
Conversely, the seeds of A. myrianthum ( Figure 6B) were 2.9-3.1 × 1.5-1.6 mm in size, characterized by subrectangular testa cells, 24.5-45.5 × 7-18.5 µm, with irregular undulate borders. The anticlinal walls were poorly detectable, appearing somehow flat to slightly channeled and covered by prominent strip-like connections forming a large intercellular region 8-12 µm wide. The periclinal walls were slightly raised, provided with 2-4 (-5) irregular and smooth verrucae arranged in a central row along the main cell axis, sometimes with some additional smaller and marginal verrucae.

Distribution and Ecology
The geographic distribution of A. sphaeronixum and its closest allied A. myrianthum and A. staticiforme is quite far from each other. While A. staticiforme is distributed in the Central Aegean Islands ( Figure 7A,B), A. myrianthum grows in southwestern Anatolia ( Figure 7C). This new species is localized near Nevşehir in Central Anatolia ( Figure 7C,D), where it grows in maquis and dwarf shrub communities, on sandy or rocky substrata, at 1000-1300 m of elevation. Conversely, A. myrianthum usually grows in grasslands linked to more or less damp soils, while A. staticiforme can be found in the clearings of coastal garrigues or maquis on various types of substrata, from sea level up to an elevation of ca. 500 m.

Conservation Status
Allium sphaeronixum is currently known only from four populations of Turkey ( Figure 7D), within an estimated area of occupancy (AOO) of 16.00 km 2 and an extent of occurrence (EOO) of 58.59 km 2 . Fairy chimneys are a unique geological formation found in the Nevşehir province of Turkey. These tall, cone-shaped rock formations are formed from soft volcanic ash and tuff, which has been eroded over time by wind and water. The harder rocks on top of the fairy chimneys protect the softer rock beneath, creating a chimney-like shape. Fairy chimneys in Nevşehir are especially famous for their historical and cultural significance. In the past, people used these chimneys as dwellings, and some were even decorated with intricate carvings and frescoes. Today, many of these chimneys have been converted into hotels and tourist attractions. There is intense tourism pressure in the region. Therefore, following the criteria established by IUCN [28], an assessment of 'Endangered' (EN, criteria A4, B1abii, iii) is suggested for the new species.

Discussion
Within the sect. Codonoprasum, Allium sphaeronixum shows closest relationships with some other East Mediterranean species belonging to the group of A. staticiforme, a species first described from the island of Kimolos (Cyclades archipelago, Greece) [29,30]. The species of this group share some very peculiar features allowing them to be well distinguished from the other taxa of the sect. Codonoprasum, so Zahariadi [31] considered it more appropriate to include them in a new section, proposed as A. sect. Phalerea Zahar. Subsequently, Brullo et al. [12] downgraded it to the rank of a subsection within the sect. Codonoprasum and typified by A. staticiforme. The most distinctive characters are represented by subglobose inflorescence and are usually dense and compact, subtended by a spathe with two rather small valves, shorter than or subequal to the inflorescence (only rarely longer), perigon white to pink, generally campanulate, small-sized (2-4 mm long), stamen filaments all or at least the inner ones exserted from the perigon, and seeds less than 4 mm long. Based on the literature [7,12,[32][33][34][35][36][37][38][39][40], in addition to A. staticiforme, many other species sharing these morphological characteristics were described from various territories of the East Mediterranean area, such as A. flexuosum d'Urv. from Astypalea island (Greece); A. myrianthum Boiss. from West Anatolia; A. wiedemannianum Regel from North Anatolia; A. phalereum Heldr. and Sartori ex Heldr. from Attica (Greece); A. weissii Boiss. from the Cyclades islands (Greece); A. exiguiflorum Hayek and Siehe from central-southern Anatolia; A. thrichocephalum Nábȇlek and A. rupicola Boiss. ex Mouterde from Lebanon; and A. nazarenum C.Brullo, Brullo Giusso, and Salmeri from Israel. Currently, many of these names are not recognized as valid distinct species and are often considered critical taxa, regarded as representing synonyms or falling into the intraspecific variability of accepted species [1,35,37,38,40].
As already highlighted, within the A. staticiforme group, A. sphaeronixum most resembles A. staticiforme (Figure 8) and A. myrianthum (Figure 9) for its gross morphology, but many diacritic features allow these species to be well distinguished. The most discriminant characteristics, listed in Table 1, mainly regard the bulb, scape size, number, and size of leaves; size and shape of flower pieces; and capsule and seeds. Specifically, A. sphaeronixum differs from the typical populations of A. staticiforme in having a bigger size; bulbs without bulbils; fewer leaves which are generally longer and wider; spathe valves unequal and much longer; stamen filaments unequal, the outer ones at first not exserted, shorter annulus; anthers often tinged with purple, shorter and rounded at the apex; ovary shortly stipitate at the base, slightly tuberculate (vs. slightly rugose) and bigger, with a much longer pinkish-white style and a bigger subglobose to globose-ovoid capsule. Other relevant differences regard the leaf cross-sections, with reference to the general outline and many anatomical features, because leaves of A. sphaeronixum show a widely fistulous mesophyll with a very limited spongy tissue (vs. filled mesophyll with widely lacunose spongy tissue), larger epidermal cells and a thinner cuticle (cf. Figure 5A), as well as the karyotype structure, as A. sphaeronixum has a little more asymmetrical chromosome complement, with one subtelocentric pair and one B-chromosome, which are missing in A. staticiforme (cf. Figures 3A,C and 4A,C). Seed coat micromorphology differs as well because A. sphaeronixum shows bigger seeds, with testa cells distinct both in size and shape (subpoligonal and nearly isodiametric vs. irregularly polygonal and subisodiametric to elongated), as well as in the micro-sculpturing patterns of both periclinal and anticlinal walls (feebly undulate borders vs. Ω-like).
Despite the general resemblance, significant differences can be clearly detected in comparison with A. myrianthum, which diverges from A. sphaeronixum due to the bigger size; green and striate scape (vs. glaucous and smooth); more leaves (5-6 vs. 3-4), which are longer but much narrower (40-50% less); bigger inflorescence (3-5 cm vs. 2.5-3 cm), with spathe valves subequal and both shorter than or as long as the inflorescence (vs. unequal, and just the smaller one shorter than inflorescence); perigon white (vs. white suffused with pinkish), with tepals unequal and oblong to linear-oblong, (vs. subequal and oblongelliptical); white stamen filaments, with anthers yellow and a bit longer; ovary stipitate long at the base, clearly tuberculate and smaller; a white and shorter style; and smaller capsule. Significant differences are also shown in the karyotype structure because, unlike the new species, A. myrianthum shows a quite homogeneous chromosome complement only consisting of metacentric pairs (cf. Figures 3A,B and 4A,B). As far as the leaf anatomy is concerned, the cross-section of A. myrianthum is clearly different in its smaller size, the semicylindrical outline with very prominent ribs (vs. subcylindrical with inconspicuous ribs), the much larger epidermal cells covered by a well-developed cuticle layer, and the lower number of vascular bundles which all have similar size (vs. numerous, with bigger sized alternating with smaller other ones). Lastly, the seed coat micromorphology further emphasizes the differences between the two species since A. myrianthum has smaller seeds, with subrectangular elongated cells (vs. isodiametric) showing a median row of 2-4 smooth verrucae on the periclinal walls (vs. one central large papillate verruca).
In addition to these differences concerning many macro-and micro-morphological, karyological, and anatomical traits, Allium sphaeronixum also diversifies in its ecology, which further supports its distinction as a new endemic species. As a matter of fact, while A. sphaeronixum grows in mountain habitats associated with maquis and dwarf shrub vegetation, A. myrianthum usually occurs in wetlands, where it is a member of marshy communities, and A. staticiforme is mainly localized in rocky coastal stands, mixed with dwarf shrub vegetation.

Morphological Study
Plant morphology of the new species was studied using living specimens collected from the locus classicus and the other known localities, cultivated 1-2 years both in the Botanical Garden of Catania (Italy) and in the Geophyte Garden in Yalova (Turkey). For taxonomic comparison, living plants of Allium myrianthum and Allium staticiforme, coming from the respective loci classici (Pamukkale in Denizli province, southwestern Turkey, and Kimolos Island in the Cyclades archipelago, South Aegean) and cultivated 1-2 years in the Botanical Garden of Catania, were examined together with several herbarium specimens (AEF, BM, CAT, FI-WEBB, G-BOISS, ISTE, and M). Qualitative and quantitative morphological features were measured and scored on at least 15 fresh samples using a Zeiss Stemi SV11 Apo stereomicroscope at 6-66× magnification. Taxonomic and morphological comparison with the most-related species was based on direct surveys from both fresh and herbarium material. The diagnostic traits of the new species and its two most allied ones are shown in Table 1. Voucher specimens of the new species are deposited in the herbaria CAT and ISTE [41].

Leaf Anatomy
The leaf anatomy was investigated on fresh material from cultivated plants, using blades of minimum-sized and maximum-sized leaves in their optimal vegetative phase from a point 3 cm above the sheaths. Samples were fixed in Carnoy solution (3:1 absolute ethanol: glacial acetic acid), then embedded in paraffin. Leaf cross-sections (ca. 10 µm thick) were double-stained with ruthenium red and light-green yellowish SF and photographed under a Zeiss Axiophot light microscope equipped with a 10 MP digital camera.

Karyology
The karyological analysis was carried out on root tips from cultivated bulbs, pre-treated with 0.3% (w/v) colchicine water solution for 3 h at room temperature, and then fixed overnight in fresh Farmer's fixative (3:1 v/v, absolute ethanol: glacial acetic acid). Root tips were hydrolyzed in 1N HCl at 60 • C for 7 min, washed, and stained with Feulgen for 1 h. Metaphase plates were analyzed and photographed under a Zeiss Axioskop2 light microscope equipped with an Axiocam MRc5 high-resolution digital camera. Karyotype parameters were evaluated from 10 well-spread metaphase plates from 5 individuals; the mean values were used for the karyotype characterization. Metaphase chromosomes were measured using the image analysis system Zeiss Axiovision 4.8, while karyotyping was performed by Cromolab© 1.1 software [42]. The chromosome types were named according to the position of the centromere: r = 1-1.3 (m) median, r = 1.3-1.7 (msm) median-submedian, r = 1.7-3 (sm), r =3-7 (st) subterminal [43,44]. All measured karyomorphometric parameters are given in Table 2. Table 2. Karyological features of A. sphaeronixum and allied species. Mean values ± standard deviation resulted from 10 good metaphase plates from different individuals of the type locality. Abbreviations: Type = chromosome nomenclature according to Levan et al. [43] and Tzanoudakis [44]; sat = satellited; TCL = total chromosome length; MCL = mean chromosome length; MAR = mean AR; MCI = mean CI; D-value = difference between total L and total S.

Seed Micromorphology
Seed test micromorphology was performed on mature, dry samples coming from herbarium specimens of type locality using a scanning electron microscope (SEM) (Zeiss EVO LS10), according to the protocol reported by Stork et al. [45], while terminology of the seed coat sculpturing followed Barthlott [46,47] and Gontcharova et al. [48].

Geographic Distribution
For the species distribution and conservation status, the GeoCAT software [49], accessed at http://geocat.kew.org/ (accessed on 12 December 2022), was used in order to calculate both the area of occupancy (AOO) and the extent of occurrence (EOO), according to the IUCN Red List criteria [28].